CircFBXW7 in patients with T-cell ALL: depletion sustains MYC and NOTCH activation and leukemia cell viability

Circular RNAs (circRNAs) are emerging as new players in leukemogenic mechanisms. In patients with T-cell Acute Lymphoblastic Leukemia (T-ALL), the recent report of a remarkable dysregulation of circRNAs incited further functional investigation. Here we focus on circFBXW7, highly expressed in T-cells, with a notably high abundance of the circular compared to linear transcript of FBXW7. Two T-ALL patient cohorts profiled with RNA-seq were analyzed in comparison with five populations of developing thymocytes as normal counterpart, quantifying circRNA and gene expression. CircFBXW7 expression was very heterogeneous in T-ALL patients allowing their stratification in two groups with low and high expression of this circRNA, not correlated with FBXW7 mutation status and T-ALL molecular subgroups. With a loss-of-function study in T-ALL in vitro, we demonstrate that circFBXW7 depletion increases leukemic cell viability and proliferation. Microarray profiling highlighted the effect of the circFBXW7 silencing on gene expression, with activation of pro-proliferative pathways, supporting a tumor suppressor role of circFBXW7 in T-ALL. Further, MYC and intracellular NOTCH1 protein levels, as well as expression of MYC target and NOTCH signaling genes were elevated after circFBXW7 depletion, suggesting an inhibitory role of circFBXW7 in these oncogenic axes. Plus, low circFBXW7 levels were associated with a particular gene expression profile in T-ALL patients, which was remarkably mirrored by the effects of circFBXW7 loss-of-function in vitro. CircFBXW7 depletion notably emerges as a new factor enhancing a proliferative phenotype and the activation of the MYC signaling pathway, key players in this aggressive malignancy. Supplementary Information The online version contains supplementary material available at 10.1186/s40164-023-00374-6.

To the Editor, CircRNAs are versatile regulators of cell biological activities and control oncogenic axes, with different mechanisms [1]. In pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL), an aggressive hematologic malignancy for which there is urgent need to identify new disease mechanisms and therapeutic targets [2], we recently unearthed circRNA dysregulation and defined circRNA signatures of molecular genetic subgroups [3], but data about the role of circRNAs are limited [3,4]. CircFBXW7, one of the most highly expressed circR-NAs in T-cells [5], is downregulated in ALL of the B-cell lineage [5] and could play oncosuppressor functions in solid tumors [6] and in acute myeloid leukemia [7].
We quantified both circRNA and gene expression from RNA-seq data (Additional file 1) in two independent T-ALL pediatric patients' cohorts (25 and 85 cases) and in sorted human thymocyte populations from healthy donors [3,8]. CircFBXW7 expression, sustained and poorly variable in normal thymocytes, was instead heterogeneous, scattered over a wide interval, in T-ALL samples (Fig. 1A). T-ALL patients with low and high circFBXW7 expression were stratified using the median value, resulting lower than the normal counterpart average. T-ALL groups with low and high circFBXW7 expression were not significantly associated with FBXW7 mutation state and T-ALL molecular subtypes with main driver genetic aberrancies (Additional file 1: Table S1,  S2).
We set up a loss-of-function study in T-ALL in vitro, choosing RPMI-8402 and ALL-SIL with high baseline expression of circFBXW7 among other cell lines (Additional file 1: Table S3 and Fig. S1). When circFBXW7 was knocked down efficiently and specifically, with FBXW7 mRNA unvaried (Fig. 1B), cell viability (MTT assay) significantly increased in both cell lines ( Fig. 1C and Additional file 1: Fig. S2). Further studies in RPMI confirmed an increase of proliferating cell rate (EdU assay; Fig. 1D). Moreover, microarray expression profiling disclosed the regulatory networks perturbed by circFBXW7 depletion, with 2265 genes significantly differentially expressed (Additional file 2: Table S4). Chemokines and pro-apoptotic genes were downregulated (Additional file 1: Fig.  S3). Cell-cycle, regulation of the transition between G1 and S phase, centrosome formation, and microtubule organization were among the most activated Gene Ontology (GO) functions (Additional file 3: Table S5; Fig. 1E), due to upregulation of pro-survival and pro-proliferative genes, positive regulators of mTOR signaling, oncogenic kinases and phosphatases. Cell-cycle regulation emerged also from dysregulated functions clustering (Fig. 1F). Two sets of MYC target genes (including NPM1, HSPE1, XPO1, USP1 and DDX18) and the NOTCH signaling pathway were among the MSigDB Hallmarks significantly upregulated upon circFBXW7 silencing (Fig. 1G, H). Western blot quantification indicated an increase in both MYC protein and NOTCH1 cleaved intracellular domain (Fig. 1I). The activation, upon circFBXW7 silencing, of MYC and NOTCH1 oncogenic axes in T-ALL could be compatible with evidence in breast cancer that circFBXW7 favors MYC degradation while stabilizing FBXW7 [6]. This effect, together with the well-known NOTCH1-dependendent MYC activation [9] suggest the hypothesis sketched in Fig. 1J of a feed forward loop converging on MYC.
The gene perturbation observed after circFBXW7 knock-down was significantly connected with gene expression signatures of FDA-approved drugs used for leukemia therapy (Fig. 1K). In line with a positive connectivity score with Dexamethasone effect, dose-response experiments showed that circFBXW7 depletion increases Dexamethasone sensitivity of T-ALL (Additional file 1: Fig. S4). Further investigation of primary samples recognized that the effects of circFBXW7 loss-of-function in vitro remarkably mirrored the condition of the patient subset with reduced levels of this circRNA. Strikingly, the genes upregulated in T-ALL cases with low circF-BXW7 expression were significantly enriched among those modulated upon its silencing in vitro, with proproliferative MsigDB Hallmarks overrepresented among the leading-edge genes commonly upregulated ( Fig. 2A). Genes significantly differentially expressed upon silencing and with concordant expression variation in both patient cohorts (Fig. 2B) were enriched in biological functions and pathways linked to cell proliferation ( Fig. 2C; Additional file 1: Fig. S5). CDK1, CDC7 and CDC25C, the YES1 oncogene, other genes associated with cell proliferation (EML4, TTK, CDCA8, NUF2, NDC80, and CENP) and stemness (IKZF2), and oncogenes targeted by MYC (XPO1 and USP1) and NOTCH1 (HES6) were commonly upregulated. Tumor suppressors (DUSP3, PRSS57 and CDIP1) were downregulated (Fig. 2B).
Our functional data of circFBXW7 silencing in T-ALL in vitro, concordantly with the recent report that its overexpression increases apoptosis in JURKAT cells [10], strongly support the concept that circFBXW7 works as a tumor suppressor in this malignancy. Of novelty, functional and patient data concordantly indicated that the abnormal depletion of circFBXW7 in T-ALL is linked with a proliferative phenotype and represents a new factor that contributes to MYC and NOTCH1 hyperactivation key for leukemia onset and progression [11]. CircFBXW7 is an additional anti oncogenic product of the gene encoding the well-known tumor suppressor protein FBXW7. FBXW7 inactivation due to somatic mutations in T-ALL resulted independent from circFBXW7 depletion, and these two molecules likely participate in an interconnected regulatory network.
CircRNAs are promising targets for innovative therapeutic strategies [12] and owing to its tumor suppressor role, circFBXW7 restoration or overexpression might be evaluated in the future to fight T-ALL.